Urinary tract infections (UTI) are among the most common serious pathogenic infections in the Western world. UTIs affect infants, children, and the elderly, but females are particularly at higher risk. Majority of UTIs are caused by Escherichia (E.) coli that expresses filamentous adhesion organelles termed type I fimbriae. The fimbriae are thought to initiate chronic UTIs by mediating adherence of E. coli to the bladder epithelium followed by invasion (or endocytosis) of the bacterium into the host cell. The intracellular invasion leads to a quiescent infection in which the bacteria are inaccessible to host-immune cells or cell-impermeable antibiotics. Emerging evidence implicates the cell membrane-associated endocytic machinery in the invasion (entry) of bacteria into the host cell. This cellular machinery, typically involved in endocytosis of membrane- bound receptors, may include caveolae and clathrin-coated pits. Significantly, both caveolae- and clathrin-coated pit-based endocytosis have been shown to be dependent upon enzymatic activity of the ubiquitous GTPase dynamin2 protein. Ability of dynamin2 to execute the fission of budding vesicles from the plasma membrane is influenced by interactions with partner proteins. Recent discoveries with purified proteins show that dynamin2 forms a complex with endothelial nitric oxide (NO) synthase (eNOS) and regulates eNOS activity. Our preliminary results demonstrate that the invasion of E. coli into bladder epithelial cells is controlled by the enzymatic activities of both dynamin2 and eNOS. The dynamin2 undergoes S-nitrosylation at specific cysteine residues and this modification is required for the E. coli invasion. The central hypothesis of this application is that eNOS and dynamin2 cooperatively regulate E. coli invasion by regulating endocytic vesicle trafficking. The associated Specific Aims are: [1] To determine how E. coli invasion into bladder epithelial cells promotes the dynamin2 S-nitrosylation with emphasis on eNOS-mediated NO production; [2] To assess effect of dynamin2 S- nitrosylation on E. coli invasion into bladder epithelial cells using in vitro bacteria invasion assays; and [3] To determine physiologic relevance of dynamin2 S-nitrosylation on E. coli invasion into bladder epithelium using animal models. The proposed studies should provide greater insight into the mechanisms involved in invasion of the uroepithelium and may ultimately identify dynamin2 S-nitrosylation as an effective drug target to limit the frequent urinary tract infections. PUBLIC HEALTH RELEVANCE: Urinary tract infections (UTIs) are among the most common and serious pathogenic infections. We identified NOS/NO and dynamin2 as regulators of E. coli invasion into bladder cells. The targeting of eNOS and dynamin2 may provide a window of opportunity to interfere with currently untreatable recurrent UTIs.